The plant-derived, diterpenoid 7-keto-sempervirol was recently reported to display moderate activity against larval stages of Schistosoma mansoni (IC = 19.1 μM) and Fasciola hepatica (IC = 17.7 μM), two related parasitic blood and liver flukes responsible for the neglected tropical diseases schistosomiasis and fascioliasis, respectively. Here, we aimed to increase the potency of 7-keto-sempervirol by total synthesis of 30 structural analogues. Subsequent screening of these new diterpenoids against juvenile and adult lifecycle stages of both parasites as well as the human HepG2 liver cell line and the bovine MDBK kidney cell line revealed structure-activity relationship trends. The most active analogue, 7d, displayed improved dual anthelmintic activity over 7-keto-sempervirol (IC ≈ 6 μM for larval blood flukes; IC ≈ 3 μM for juvenile liver flukes) and moderate selectivity (SI ≈ 4-5 for blood flukes, 8-13 for liver flukes compared to HepG2 and MDBK cells, respectively). Phenotypic studies using scanning electron microscopy revealed substantial tegumental alterations in both helminth species, supporting the hypothesis that the parasite surface is one of the main targets of this family of molecules. Further modifications of 7d could lead to greater potency and selectivity metrics resulting in a new class of broad-spectrum anthelmintic.

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Teucrium yemense (Defl), locally known as Reehal Fatima, is a medicinal plant commonly grown in Saudi Arabia and Yemen. Phytochemical investigation of the aerial parts of T. yemense yielded six new neoclerodane diterpenoids, namely fatimanol A-E (1, 2, 3, 5, and 6) and fatimanone (4), and the known teulepicephin (7). As both the Teucrium genus and the related Lamiaceae family have previously been widely reported to possess anthelmintic and antimicrobial activities, the structural and biological characterization of the seven diterpenoids was pursued. The structures of the new compounds were elucidated from their 2D NMR and MS profiles and by comparison to related compounds. The structure of fatimanol D (5) was confirmed by X-ray crystallographic analysis. The new structures contribute to the breadth of knowledge of secondary metabolites in this genus.

BackgroundPraziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium.Methodology/Principle findingsThirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 μM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (cell impermeable inhibitor).Conclusion/SignificanceCollectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.

29 2 30 Abstract 31 32 Background33 Praziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected 34 tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While 35 this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often 36 requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-37 schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning 38 strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the 39 Structural Genomics Consortium. 40 41 Methodology/Principle findings 42 Thirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially 43 screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm 44 platform. At 10 µM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and 45 phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against 46 schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), 47 one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-48 507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone 49 demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for 50 schistosome development, further characterisation of these compounds/putative targets was 51 performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target 52 (Smp_000700) in adult worms replicated the compound-mediated motility and egg production 53 defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced 54 by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition 55 and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 56 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue 57 GSK-J1 (non-permeable inhibitor).58 3 59 Conclusion/Significance 60 Collectively, these results provide further support for the development of next-generation drugs 61 targeting schistosome epigenetic pathway components. In particular, the progression of histone 62 methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control. 63 64 65 4 66 Author Summary 67 Human schistosomiasis is caused by infection with parasitic blood fluke worms. Global control of 68 this NTD is currently facilitated by administration of a single drug, praziquantel (PZQ). This mono-69 chemotherapeutic strategy of schistosomiasis control presents challenges as PZQ is not active 70 against all human-dwelling schistosome lifecycle stages and the evolution of PZQ resistant parasites 71 remains a theat. Therefore, new drugs to be used in combination with or in replacement of PZQ are 72 urgently nee...